Hereditary multiple exostoses (HME or EXT) is an autosomal dominant disorder characterized by multiple exostoses most commonly arising from the juxtaepiphyseal region of the long bones. Other bones that can be involved include the pectoral and pelvic girdles, rib, and less frequently, vertebrae, sternum, skull, and carpal and tarsal bones. In addition, abnormal bone modeling, particularly of the long ones, is a feature. This causes bowing, shortness, cortical irregularities, and metaphyseal widening of the involved bones, leading to the deformities of the forearms and disproportionate short stature in severe cases. The exostoses can give rise to complications such as compression or irritation of adjacent nerves, vessels, and tendons, and urinary or intestinal obstruction. The most serious complication is sarcomatous degeneration, which occurs in 0.5 to 2% of affected individuals (Hennekam, R. C. M., Hereditary Multiple Exostoses, J. Med. Genet. 28:262-266, (1991)). Hecht et al. (Hecht, J. T.; Hogue, D.; Strong, L. C.; Hansen, M. F.; Blanton, S. H.; Wagner, M., Hereditary Multiple Exostosis and Chondrosarcoma: Linkage to Chromosome 11 and Loss of Heterozygosity for EXT-Linked Markers on Chromosomes 11 and 8., Am. J Hum. Genet. 56:1125-1131 (1995)) reported that a prevalence of chondrosarcoma in multiple exostoses of 2% to 5% compared with age-adjusted incidence rate of 1/100,000 for all bone cancers. Multiple exostoses are part of the Langer-Giedion syndrome (150230), which appears to be a contiguous gene syndrome, due to deletion in the 8q24 region.
Genetic linkage studies established three loci for multiple exostosis genes. They are located at chromosome 8, 11 and 19, respectively.
Cook et al. (Cook, A.; Raskind, W.; Blanton, S. H.; Pauli, R. M.; Gregg, R. G.; Francomano, C. A.; Puffenberger, E.; Conrad, E. U.; Schmale, G.; Schellenberg, G.; Wijsman, E.; Hecht, J. T.; Wells, D.; Wagner, M. J., Genetic Heterogeneity in Families with Hereditary Multiple Exostoses., Am. J Hun. Genet. 53:71-79 (1993)) concluded that about 70% of multiple exostoses families show linkage to markers in the 8q24. 11-q24.13 region. Investigating 2 large exostoses pedigrees in which linkage to markers from 8q24 was excluded, Wu et al. (Wu, Y.-Q.; Heutink, P.; de Vries, B. B. A.; Sandkuijl, L. A.; van den Ouweland, A. M. W.; Niermeijer, M. F.; Galjaard. H.; Reyniers, E.; Willems, P. J.; Halley, D. J. J., Assignment of a Second Locus for Multiple Exostoses to the Pericentromeric Region of Chromosome 11, Hum. Molec. Genet. 3:167-171 (1994)) found evidence of linkage to microsatellite markers from the proximal short and long arms of chromosome 11. The highest lod score by 2-point analysis was found with D11S554; maximum lod=7.148 at theta=0.03.
Hecht et al. (1995) and Raskind et al. (Raskind, W. H.; Conrad, E. U.; Chansky, H.; Matsushita, M., Loss of Heterozygosity in Chondrosarcomas for Markers Linked to Hereditary Multiple Exostoses Loci on Chromosomes 8 and 11, Am. J Hum. Genet. 56:1132-1139 (1995)) presented evidence suggesting that the EXT1 gene on chromosome 8 and the EXT2 gene on chromosome 11 have tumor-suppressor function. They found loss of heterozygosity for markers linked to these 2 genes in chondrosarcomas originating in individuals with multiple exostoses and in sporadic chondrosarcomas.
As part of a larger study to determine the frequency of the 3 EXT types in the United States, Hecht et al. (1995) ascertained a large multigenerational family with multiple exostosis and in 1 member a chondrosarcoma. This family demonstrated linkage of the disease to chromosome 11 markers. Constitutional and tumor DNAs from the affected family member were compared using short-tandem-repeat (STR) markers from chromosomes 8, 11, and 19. Loss of heterozygosity (LOH) in the tumor was observed for chromosome 8 and 11 markers, but chromosome 19 markers were intact. Hecht et al. (1995) observed an apparent deletion of D11S903 in constitutional DNA from all affected individuals and in the tumor sample. These results indicated that EXT2 gene maps to the region containing D11S903, which is flanked by D11S1355 and D11S1361. The authors similarly analyzed additional constitutional and chondrosarcoma DNA from 6 unrelated individuals, 2 of whom had EXT. One tumor from an individual with EXT demonstrated LOH for chromosome 8 markers, and a person with a sporadic chondrosarcoma was found to have a tumor-specific LOH and a homozygous deletion of chromosome 11 markers. These findings suggested to Hecht et al. (1995) that EXT genes may be tumor-suppressor genes and that the initiation of tumor development may follow a multistep model. Raskind et al. (1995) found loss of constitutional heterozygosity at polymorphic loci linked to genes involved in the multiple exostoses. They detected LOH for markers linked to EXT1 on chromosome 8 in a chondrosarcoma that arose in a man with multiple exostoses. They also found LOH for markers linked to EXT1 in 4 of 17 sporadic chondrosarcomas, and 7 showed LOH for markers linked to EXT2. In all, Raskind et al. (1995) observed LOH for markers linked to EXT 1 or EXT2 in 44% of the 18 tumors, whereas heterozygosity was retained for markers on 19p linked to EXT3. These findings also suggested a tumor suppressor role for EXT genes.
Studying 7 extended multiple exostoses families, all linked to the EXT2 locus, Wuyts et al. (Wuyts, W.; Ramlakhan, S.; Van Hul, W.; Hecht, J. T.; van den Ouweland, A. M. W.; Raskind, W. H.; Hofstede, F. C. Reyniers, E.; Wells, D. E.; de Vries, B.; Conrad, E. U.; Hill, A.; Zalatayev, D.; Weissenbach, J.; Wagner, M. J.; Bakker, E.; Halley, D. J. J.; Willems, P. J., Refinement of the Multiple Exostoses Locus (EXT2) to a 3-cM Interval on Chromosome 11, Am J. Hum. Genet. 57: 382-387 (1995)) refined the localization of the EXT2 gene to a 3-cM region flanked by D11S1355 and D11S1361/D11S554. The findings indicated that the EXT2 gene is located on 11p12-p11. The refined localization excluded a number of putative candidate genes located in the pericentromeric region of chromosome 11.
Support for the localization of the EXT2 gene was provided also by the report of McGaughran et al. (McGaughran, J. M.; Ward, H. B.; Evans, D. G. R., WAGR Syndrome and Multiple Exostoses in a Patient with Del(11)(p11.2p14.2), J. Med. Genet. 32: 823-824, (1995)), who described a patient with the combination of multiple exostoses with the WAGR syndrome (Wilms tumor, aniridia, genital anomalies, and mental retardation; 194070), a well documented contiguous gene syndrome resulting from deletion of 11p13. Their patient showed adel(11) (p14.2p11.2).
As pointed out by Potocki et al. (Potocki, L.; Greenberg, F.; Shaffer, L. G., Interstitial Deletion of 11(p12p11.2): A Rare Chromosomal Syndrome with Mental Retardation, Parietal Foramina, and Multiple exostoses. (Abstract), Am. J Hum. Genet. 57: A123 (1995)) the description of the contiguous gene syndrome resulting from interstitial deletion of 11p, del(11)(p12p11.2), including multiple exostoses as a feature, provided confirmation of the mapping of EXT2. Other features of this contiguous gene syndrome are mental retardation and parietal foramina, known as Catlin marks. Potocki and Shaffer (Potocki, L.; Shaffer, L. G., Interstitial Deletion of 11(p11.2p12): a Newly Described Contiguous Gene Deletion Syndrome Involving the Gene for Hereditary Multiple Exostoses (EXT2), Am. J Med. Genet. 62:319-325 (1996)) reported the clinical and molecular findings in a further patient with an 11(p12p11.2) deletion. Cytogenetic and molecular analysis demonstrated a de novo, paternally-derived deletion for markers known to be tightly linked to EXT2. The patient had an unusual facies (bilateral epicanthal folds, ptosis, short philtrum, and downturned upper lip), mental retardation, multiple exostoses, brachycephaly, and bilateral parietal foramina Ahn et al. (Ahn, J. et al., Cloning of the Putative Tumor Suppressor Gene for Hereditary Multiple Exostoses, Nature Genet., 11, 137-143 (1995)) cloned and characterized a cDNA that spans chromosomal breakpoints previously identified in 2 multiple exostoses patients. Furthermore, the gene harbored frameshift mutations in affected members of 2 EXT1 families. The cDNA had a coding region of 2,238bp with no apparent homology to other known gene sequences.
Stickens et al. (Stickens, D.; Clines, G.; Burbee, D.; Ramos, P.; Thomas, S.; Hogue, D.; Hecht, J. T.; Lovett, M.; Evans, G. A., The EXT2 Multiple Exostoses Gene Defines a Family of Putative Tumour Suppressor Genes, Nature Genet. 14:25-32 (1996)) reported the identification and characterization of the EXT2 gene on chromosome 11. The EXT2 gene contains 7 exons and encodes a 718-amino acid polypeptide. By Northern analysis, they detected a 3.5-kb transcript in almost all tissues. Alternative splicing generates a mninor 3.7-kb transcript in some tissues. The gene shows striking sequence similarity to the EXT1 gene on chromosome 8. Multigenerational family with chromosome 11-linked multiple exostoses described by Hecht et al. (1995) in which one family member developed a malignant chondrosarcoma. In this family, an apparent deletion of the region including polymorphic marker D11S903 was observed in all affected members. In tumor tissue derived from the patient with chondrosarcoma, Stickens et al. (1996) observed that polymorphic marker D11S903 was deleted and that there was LOH for other chromosome 11 markers. In the initial study of the EXT2 candidate gene for mutations, they performed SSCP analysis using DNA from affected members of 3 unrelated families. In 1 of 2 alleles of 1 patient, deletion of nucleotides 784 to 787 was identified, resulting in a frameshift and a premature termination of the EXT2 gene product.
Using genoric DNA library constructed from 11p11-12 by microdissection, several cDNA clones from human placenta cDNA library were isolated. One of these clones shares 59% homology with EXT1 cDNA in some parts. This gene was further mapped by fluorescence in situ hybridization to chromosome 11p11, which is the exact region where EXT2 gene is located. By cDNA library screening and 5'RACE techniques, the full cDNA sequence was cloned. It is predicted that this cDNA encodes 728 amino acids. Both cDNA and amino acids sequence share striking similarities with EXT1. Thus, this gene is responsible for chromosome 11 linked multiple exostosis.
By comparing the cDNA sequence of the present invention with the reported EXT2 gene sequence, they are 90bp difference in the middle of the coding region which results in 30 amino acids difference from the reported one by Stickens (Stickens, 1996).
Stickens et al isolated this cDNA from brain cDNA library, while the present cDNA is from placenta cDNA library. The difference between our DNA sequence and theirs indicates that there are at least two isoforms of EXT2-encoded proteins.
This indicates that EXT2 genes are useful as therapeutic targets. Clearly there is a need for identification and characterization of further genes which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, hereditary multiple exotoses and cancers, such as chondrosarcoma, among others.